The goal of this project is to develop nanocapillary electrophoresis array technology (NEAT) as a label-free, quantitative, and high-throughput assay of proteins and nucleic acids that are critical in early detection, monitoring, diagnosis, and prognostic evaluation of cancer. The NEAT chips will contain an array of glass nanocapillaries whose inner surfaces are functionalized with receptors to capture specific ligands. We have developed a process for synthesizing glass nanocapillaries with lengths in the 1-10 micron range and diameters in the 5-20 nm range, which is on the order of biomolecular size. Hence, when analyte biomolecules are electrophoretically transported through functionalized nanocapillaries in response to an applied voltage (approx. 1 V), specific ligand-receptor binding will lead to reduction in the ionic current flow due to partial blockage of the nanocapillary. Recent experiments have shown that indeed when a transmembrane ion channel protein is functionalized with a single strand of DNA, binding of its complementary strand can be detected through modulation of the ionic current with single-molecule sensitivity, and specificity of single base pair mismatches. By using the same principles, the goal of the R21 phase is to demonstrate that ionic current modulation could be used as a label-free assay for PSA protein and p53 exonic sequence with specificity and sensitivity sufficient for cancer diagnostics and monitoring. The goal of the R33 phase is to build upon the R21 knowledge and develop the NEAT chip that will consist of an array of microfluidic cells, with each cell containing a single nanocapillary functionalized with a distinct probe/receptor. An electronic system will allow multiplexed addressing of individual nanocapillaries such that their respective ionic currents can be simultaneously measured. Such an integrated system will enable multiplexed assays of literally hundreds of molecules, such as transcribed mRNAs or proteins. For protein expression, in particular, this technology would represent a new paradigm in the evaluation of multiple proteins from a single tissue or from serum and could represent a cost-effective way to assess multiple cancer antigens in cancer screening and monitoring programs. Such routine screening is now only done for very few cancer antigens (primarily prostate specific antigen, PSA) due to the expense such large-scale screening would incur. This technology would directly address this important public health issue.